Kondo Machiko, Hirai Hiroyuki, Furukawa Takehito, Yoshida Yuki, Suzuki Aika, Kawaguchi Takemasa, Che Fang-Sik
Graduate School of Bioscience, Plant Molecular Physiology, Nagahama Institute of Bio-Science and Technology Nagahama, Japan.
Front Plant Sci. 2017 Jan 4;7:1988. doi: 10.3389/fpls.2016.01988. eCollection 2016.
Many plant pathogens inject type III (T3SS) effectors into host cells to suppress host immunity and promote successful infection. The bacterial pathogen causes brown stripe symptom in many species of monocotyledonous plants; however, individual strains of each pathogen infect only one host species. T3SS-deleted mutants of K1 (virulent to rice) or N1141 (virulent to finger millet) caused no symptom in each host plant, suggesting that T3SS effectors are involved in the symptom formation. To identify T3SS effectors as virulence factors, we performed whole-genome and predictive analyses. Although the nucleotide sequence of the novel leucine-rich repeat protein () gene of N1141 had high sequence identity with K1 Lrp, the amino acid sequences of the encoded proteins were quite different due to a 1-bp insertion within the K1 gene. An Lrp-deleted K1 strain () did not cause brown stripe symptom in rice (host plant for K1); by contrast, the analogous mutation in N1141 () did not interfere with infection of finger millet. In addition, retained the ability to induce effector-triggered immunity (ETI), including hypersensitive response cell death and expression of ETI-related genes. These data indicated that K1 Lrp functions as a virulence factor in rice, whereas N1141 Lrp does not play a similar role in finger millet. Yeast two-hybrid screening revealed that K1 Lrp interacts with oryzain α, a pathogenesis-related protein of the cysteine protease family, whereas N1141 Lrp, which contains LRR domains, does not. This specific interaction between K1 Lrp and oryzain α was confirmed by Bimolecular fluorescence complementation assay in rice cells. Thus, K1 Lrp protein may have acquired its function as virulence factor in rice due to a frameshift mutation.
许多植物病原体将III型（T3SS）效应蛋白注入宿主细胞，以抑制宿主免疫并促进成功感染。这种细菌病原体在许多单子叶植物物种中引起褐色条纹症状；然而，每种病原体的个别菌株仅感染一种宿主物种。K1（对水稻致病）或N1141（对黍致病）的T3SS缺失突变体在各自的宿主植物中均未引起症状，这表明T3SS效应蛋白参与了症状形成。为了鉴定T3SS效应蛋白作为致病因子，我们进行了全基因组和预测分析。尽管N1141的新型富含亮氨酸重复蛋白（Lrp）基因的核苷酸序列与K1 Lrp具有高度序列同一性，但由于K1 Lrp基因内有一个1碱基对的插入，编码蛋白的氨基酸序列有很大差异。缺失Lrp的K1菌株（K1ΔLrp）在水稻（K1的宿主植物）中未引起褐色条纹症状；相比之下，N1141（N1141ΔLrp）中的类似突变并不影响对黍的感染。此外，K1ΔLrp保留了诱导效应蛋白触发免疫（ETI）的能力，包括过敏反应细胞死亡和ETI相关基因的表达。这些数据表明，K1 Lrp在水稻中作为致病因子发挥作用，而N1141 Lrp在黍中不发挥类似作用。酵母双杂交筛选显示，K1 Lrp与水稻半胱氨酸蛋白酶家族的病程相关蛋白oryzain α相互作用，而含有LRR结构域的N1141 Lrp则不与之相互作用。K1 Lrp与oryzain α之间的这种特异性相互作用在水稻细胞中通过双分子荧光互补试验得到了证实。因此，由于移码突变，K1 Lrp蛋白可能在水稻中获得了其作为致病因子的功能。
